In order to understand the development and functioning of the thymus, both in terms of T cell differentiation and stromal cell environmental support, we have undertaken a molecular approach to identify genes that are uniquely expressed in this organ. We have created a RT-PCR based subtracted cDNA library from fetal thymic stromal cells. The novel cDNAs were then screened for expression in various tissues and in a set of SV40 transformed thymic epithelial cell lines. Multiple full length cDNA clones were isolated from a SCID thymus library. The three novel genes were named Epithin, Spatial, and Thymic Stroma Co-Transporter (TSCOT). We are currently focusing on two of these gene products (TSCOT and Epithin) during the past year. 1). The lab has been making good progress in generating model mouse systems which show a thymus-specific expression pattern of targeting of exogenous genes specifically in different thymic epithelial cell compartments using transgenic and knock-in mouse approaches. A transgenic mouse line containing 3.1kb of the TSCOT promoter driving Enhanced Green Fluorescent Protein (EGFP) was established to identify the thymic stromal cell lineage in which TSCOT is expressed. Surprisingly, this mouse showed EGFP expression in the subcapsular and medullary compartments of the thymus in contrast to the cortex where the endogenous gene is expressed. In another model, a mouse was generated by knocking in the LacZ gene at the TSCOT locus. We have shown that LacZ is expressed only in the thymic epithelial cell compartment. Interestingly, expression of these targeted proteins changes during development. Using this information, we are now generating more mouse lines which can result in specific cell ablation in a constitutive or inducible fashion in the different thymic stromal cell compartments. 2). In collaboraion with Dr. D. Park, we have been successful in identifying proteins involved in interactions with Epithin using Yeast 2 hybrid technology. In addition, we are continuing our search for the general nature of Epithin's function and the mechanism of its processing during cellular signaling .